Gene silencing events associated with promoter region methylation are common in malignant cells. An unresolved issue for these silencing events is whether promoter region methylation represents the beginning or end of the process. The major goal of this application is to use mouse Aprt to demonstrate that promoter region methylation is the endpoint of a multistep silencing process. Specifically, methyl binding proteins (MBPs), which are known to bind to methylated regions within the genome and to attract repressive complexes, will be used to trigger silencing at a distance from the promoter. Additional experiments will demonstrate that the promoter has a significant, yet imperfect, mechanism to block MBP-induced silencing and that selective processes are sufficient to maintain silencing. Finally, it will be shown that promoter region methylation is the inevitable endpoint of the silencing process, and that this late event serves to stabilize chromatin changes that accompany MBP-mediated silencing at distance. The demonstration of each step of a multistep silencing pathway, which is currently undefined, will provide novel insight into this aberrant process in malignant cells.